William Bloom

Histological Changes Following Radiation Exposures

Our group, which consisted of Drs. Murray, DeBruyn, Pierce, and Snider, Miss Rhoades, Miss Heller, my wife, and, for part of the time, Drs. Block, Conway, Jacobson, and Lisco, examined large series of animals, exposed to a variety of external and internal sources of radiation: x-rays; fast and slow neutrons; gamma rays from radium and from the Clinton pile; beta rays from the external phosphorus source mentioned previously in this Syrnposium, and internally administered radium, plutonium, sodium24, phosphorus32, barium140, lathanum140, strontium89, radio zirconium, yttrium91, and in a few instances radio cerium. The work was done under the general direction of Dr. R. S. Stone and was carried out in the laboratories supervised by Dr. Kenneth S. Cole in Chicago and Dr. Howard J. Curtis at Clinton Laboratories. Most of our experiments were stereotyped. Animals were given the ld50/30-days dose and fractions thereof with the hope that we would find the lower limit of irradiation damage as shown by the microsco...

A TEXTBOOK OF HISTOLOGY

The cell Epithelium Glands and secretion Blood Connective tissue proper Adipose tissue Cartilage Bone Bone marrow and blood cell formation Muscular tissue The nervous tissue, Jay B. Angevine Blood and lymph vascular systems The immune system, Elio Raviola Thymus, Elio Raviola Lymph nodes, Elio Raviola Spleen, Elio Raviola Hypophysis The thyroid gland Parathyroid glands Adrenal glands and paraganglia Pineal gland Skin Oral cavity and associated glands The teeth The oesophagus and stomach Intestines The liver and gall-bladder Pancreas Respiratory system The urinary system Male reproductive system Female reproductive system Mammary gland The eye The ear.

Transformation of Lymphocytes of Thoracic Duct into Polyblasts (Macrophages) in Tissue Culture.

The ability of the lymphocytes of the blood stream to develop into either myeloid or phagocytic cells is denied by most investigators. In a long series of experiments Maximow 1 showed that the lymphocytes and monocytes of the blood wander into the tissues in inflammation and transform there in a very short time into exudate mononuclear cells or polyblasts, as he called them. A portion of the latter he also derived from his resting wandering cells or histiocytes. These, as he showed, have a close embryogenetic relationship to the lymphocytes. Several investigators have studied the development of lymphocytes and lymphoid tissue in tissue culture. Their results have not been uniform or decisive because it was very difficult to follow the fate of the various kinds of cells present in the explanted tissues. At the suggestion, and with the aid of Dr. A. Maximow, I cultivated the lymph of the thoracic duct of the rabbit. This fluid in the rabbit contains only lymphocytes, or at most an occasional monocyte. (Simpson, 2 Thorne and Evans, 3 Kindwall. 4 ) Lymph was pipetted aseptically from the cervical portion of the thoracic duct of adult rabbits (anesthetized with ether) which had been starved for 24 hours. The lymph was allowed to clot; it was then minced in Ringers solution and explanted in heparin plasma with various tissue extracts and tissues. This report gives the results obtained in cultivating lymph with embryonic extract. Except for a few scattered monocytoid cells and erythrocytes, the lymph used in these experiments, as seen in spravital, dry and wet smears, contained only lymphocytes. After the cultures were studied in the living condition, both with and without supravital staining with neutral red, they were fixed in Zenker-formol, imbedded in celloidin, cut in serial sections, and stained with hematoxylin-eosin-azure II.

CHANGES IN FINE STRUCTURE OF PARTS OF CHROMOSOMES AFTER LOCALIZED ULTRAVIOLET IRRADIATION

A segment of a chromosome after being irradiated with the Uretz ultraviolet microbeam1s2 undergoes a prompt change in refractive index and appears optically negative instead of black with dark contrast phase micro~copy.~ For convenience we have often called this phenomenon “paling,” but this is a poor term since the changed area would undergo “darkening” when examined with a bright contrast phase microscope. When the cells are fixed for cytological study, the locally irradiated areas do not stain with most nuclear stains or with methyl green in the pyroninmethyl green mixture, and react very faintly or not a t all with the Feulgen method? Further study has shown that if sufficient time is allowed to elapse between irradiation and fixation (45 or 60 min.) the irradiated areas become completely Feulgen negative. Ultraviolet absorption studies show that the irradiated areas have a greatly diminished absorption a t 2600 A . 5 The failure of the irradiated area to stain with the Feulgen and pyroninmethyl green methods and the decreased absorption a t 2600 A suggest that the nucleic acid had been greatly changed or lost from this portion of the chromosome. From the change in refractive index in the area one might suspect that it had lost some material. This inference is bolstered with preliminary interference microscopy measurements of optical path differences between irradiated and nonirradiated chromosomal regions that suggest a relative dry mass loss of about 15 per cent from the former (R. H. Haynes and M. Stodolsky, personal communication). For the last two years I have been studying the structure of the changed areas in the chromosomes with the electron microscope. To this end a convenient method was devised by which the partially irradiated cell could be found and sectioned for electron microscopyP The somatic cells studied were mesothelial cells growing in an epithelial sheet from explants of urodele heart in chick plasma diluted 1 to 9 in amphibian Tyrode solution. Some of the findings with the electron microscope may be summarized as follows. I n cells fixed with 1 or 2 per cent osmic acid solutions a t several hydrogen ion concentrations I have not been able to find structural differences between the chromosomes in the irradiated and nonirradiated areas. In all of them the chromosomes appear as masses of short rods or of tiny black dots that sometimes form short chains; the spaces between the rods and dots are very pale and do not suggest any structure. The outlines of the chromosomes are exceedingly vague, and it is difficult to tell where the chromosomes end. After several months spent with these negative experiments, I began to study the living cells with dark contrast phase microscopy during fixation. The first significant finding was that the pale, irradiated area, clearly visible as a bright zone with phase microscopy, disappeared as soon as osmic vapor

Relative Toxicity of Antiseptics on Bacteria and Tissues in Cultures.

Lambert, 1 Lambert and Meyer, 2 and German 3 believe that an ideal antiseptic is one which kills the bacteria without harming the tissue cells and they have accordingly devised methods of comparing the toxic effects of antiseptics upon bacteria and tissue cells in vitro. Lambert after exposing fragments of connective tissue for several minutes to a saline suspension of Staphylococcus aureus transferred them into various concentrations of mercuric chloride, potassium mercuric iodide, potassium cyanide, Dakins solution, iodine, phenol, tricresol, argyrol, hydrogen peroxide, glycerol, or alcohol. After one hour the fragments were transferred to physiological salt solution where they remained from a few minutes to half an hour until explanted. Although most of the antiseptics killed the tissue cells at concentrations that did not kill the bacteria, a good growth of cells was observed after exposure for one hour to a 1:2,000 iodine solution, a strength sufficient to sterilize the tissue completely in most instances. Lambert and Meyer placed rabbit spleen fragments in a Staphylococcus aureus suspension for one minute, and then for 20 minutes in various concentrations of alcohol, iodine, mercuric chloride, mercurochrome, acriflavin, protargol, albargin, gentian violet, neo-salvarsan, or hexylresorcinol. The fragments were then washed twice in salt solution. The best results were obtained with iodine, mercuric chloride, and neosalvarsan. It is possible that in these cultures the cells may have come from the center of the explant where they were partially protected. In a second series they added the antiseptics directly to the culture medium. In these cultures higher dilutions were used, and neosalvarsan alone was less toxic for cells than bacteria, i. e. inhibiting bacteria in dilutions of 1:10,000 to 1:10,10 while cells were only slightly damaged by these dilutions. They concluded that iodine and mercuric chloride are nearer the ideal than some of the newer preparations.

Development of Elastic Fibers in Cultures of Embryonic Heart Muscle.

Because of the close association of elastic fibers with contractile tissues throughout the body, cultures of embryonic heart muscle were tested for the presence of elastic fibers. I have been unable to find a report of such an experiment. Portions of the heart of an 8 cm. guinea pig embryo and a 4 cm. rabbit embryo were cultured in homogenous embryonic extract and plasma. The cultures were made on round coverslips by Maximows method and in Carrel flasks so that in no case was it necessary to cut them for transplantation. They were fixed after 10-17 days with one or more transplantations in Zenker-formol, embedded in colloidin and serially sectioned. They were stained with Weigerts resorcin-fuchsin method for elastic fibers and counter-stained with azo-carmine. Some of the sections were stained by Foots method for reticulum fibrils. Typical elastic fibers develop regularly in those cultures in which the muscle was contracting up to the time of fixation. They appear in the zone of migrating newly formed cells outside the explanted bits of heart muscle. They are thin, blue-black lines which are fairly straight and parallel to the long axis of the migrating spindle shaped cells. The fibers branch occasionally and seem to be definitely intercellular. In some places they are curled and twisted and form bizarre figures of many shapes. In a few areas the newly formed fibers interlace and form a wide or narrow meshed network. Control sections of the hearts from which these cultures were made show that the only elastic fibers in them are in the walls of the relatively small cardiac vessels. In these experiments, elastic fibers have been produced for the first time in tissue culture.

A Textbook of Histology

Professor Maximow has left his mark in medical history as a cytologist of the first rank, and it is therefore fortunate that he should have left behind him when he died sullicient manuscript and notes to enable a book on histology to be published in his name. The historical value of such a book would in any case be considerable, but it is greatly to the credit of the second author that this book has proved of such practical value as well, that another edition was demanded within four years. We are not at all surprised that this book should have proved so popular, as it is in every way a satisfactory one. The plan of arrangement usual in a book of this kind is followed. The descriptions are clear and the illustrations well chosen; the latter maintain a nice balance between realism and clarity, and the half-dozen coloured plates are especially useful. The sections on the blood, on the blood-forming and destroying tissues, and on the spleen claimed most of the reviewers attention. These sections are particularly good, though emphasis is usually laid on the theory favoured by the late Professor Maximow. For example, the present author is very hard on all the pluralist theories of blood formation, and one would gather from reading these sections that the unitarian theory of the common origin of all blood elements from a single stem cell, the ha3inocytoblast,

Local and Generalized Defense Reactions in Animals

I have tried to show that a great variety of stimuli are constantly acting on the animal body and that diverse mechanisms act to incorporate them into the functioning organism. If for reasons of quantity or quality, the mechanisms reacting to these stimuli are inadequate, injury results. In the gradation between physiological and pathological reactions, I have set an arbitrary limit based on the criterion of whether the mechanisms immediately available at the site of action of the stimulus are adequate to neutralize the stimulus. The action of many types of stimuli results in local injury and the immediate reaction of the animal is by inflammation. Through this non-specific reaction, blood plasma and large numbers of phagocytes accumulate in the injured area. The blood contributes the vast majority of these through its granulocytes-which are relatively specialized phagocytes-and through its lymphocytes and monocytes which soon hypertrophy into macrophages. The phagocytes remove or neutralize much of the foreign material. The simple inflammatory reaction is often an incomplete barrier to the extension of the harmful agent. As a consequence, further defense mechanisms of more generalized type become active. If the injurious agents are foreign proteins or parasites, specific antibodies are formed against them-presumably by the macrophages-as part of the process of immunization. Simple inflammation differs from immune inflammation, for in the latter case the organism through its natural or acquired immunity reacts specifically and in most instances very efficiently in the neutralization of noxious factors. The generalized reintroduction of a foreign protein or parasite into an animal which is specifically immunized results in most instances in a relatively quick neutralization of the noxious agent through the efficient interaction of macrophages and antibodies. In many cases, particularly if the dosage of the stimulus is small, these reactions may be so efficient that it may be difficult to demonstrate the reactivity of the host. Here it is apparent that a stimulus, which in a non-prepared host would have required an extensive series of defense mechanisms, is now overcome by the first line of defense of the immunized animal. The lymphatic tissue acts as a mesenchymal reserve in the simple and immune, local and generalized reactions by furnishing large numbers of lymphocytes which readily turn into macrophages.

A Text-Book of Histology

1 Automatic temperature compensating control. No water or other cooling devices necessary. Eliminates difficulty such as cloudiness caused by vapors or moisture. 2 Regulation of light output by means of rheostat control so that the current output of the cells is definitely known. 3 Photoelectric cells balanced by an ultra-sensitive galvanometer. 4 Milliammeter indicates output of photocells. Sensitive to slightest fluctuation of the electric circuit. 5 Percentage readings from 0 to 100 in 1/lOths. Detailed Information in Builletin P 546

An Immune Reaction in Tissue Culture.

The method of tissue culture has not been extensively used for immunological purposes. Carrel and Ingelbritsten 1 and Przygode 2 have shown that lymph node, bone marrow and spleen react in tissue culture to the presence of antigens by the formation of specific antibodies. This report is the first on a series of experiments in which we have cultivated various tissues from normal and immunized animals and have watched the effect of the addition of antigens to the cultures. In one set of experiments we cultured the lungs of 8 normal rabbits and those of 5 rabbits immunized against pigeon erythrocytes. After allowing the cultures to develop for several days, they were washed in Ringers solution. Each culture then received a drop of pigeon erythrocytes suspended in bone marrow or embryonic extract. The cultures were carefully observed in the living condition and after fixation at various periods were sectioned and stained. Cultures of rabbit lung are characterized by great numbers of phagocytes; these we consider to be of mesenchymal origin. They are identical with the alveolar phagocytes of the adult lung. Pigeon erythrocytes when added to cultures of lungs from normal rabbits attach themselves to the phagocytes of the cultures but are rarely taken up by these cells in periods as long as twelve days. The phagocytes in cultures of lungs from immunized rabbits, however, take up great numbers of the erythrocytes in a few hours. No difference was noted in the reactions of the lungs from those rabbits which had previously received one injection, and those which had received two injections of pigeon erythrocytes in the process of immunization.